Dialuminium zinc tetraoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dialuminium zinc tetraoxide

EC Number:

235-101-0

EC Name:

Dialuminium zinc tetraoxide

Cas Number:

12068-53-0

Molecular formula:

ZnAl2xO(3x+1), x = 1 – 9

IUPAC Name:

dialuminum; zinc(2+); oxygen(2-)

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): D-PrisM
- Physical state: pale, yellowish powder
- Analytical purity: 100%
- Composition of test material: zinc aluminate
- Lot/batch No.: PC080519/1
- Expiration date of the lot/batch: June 2018
- Stability under test conditions: stable at room temperature
- Storage condition of test material: in a closed vessel at room temperature protected from humidity

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment: 55, 150, 499, 1508 and 5005 µg/plate with and without metabolic activation
Second experiment: 324, 625, 1247, 2527 and 5003 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine (20 µg in DMSO, -S9, TA 97a, TA 98 and TA 102); sodium azide (1 µg in water; -S9; TA 100 and TA 1535); 2-Amino-anthracene (1 µg in DMSO, +S9; TA 97a, TA 100, TA 102 and TA 1535); Benzo-a-pyrene (20 µg in DMSO; + S9, TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: pre-incubation

DURATION
- Preincubation period: 20 min (second experiment)
- Exposure duration: 48 h (first and second experiment)

NUMBER OF REPLICATIONS: 4 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity was evaluated at test concentrations of 5007 and 1504 µg/plate. Per tester strain, 4 plates were exposed for 48 h in a plate incorporation test. The quotient of the number of revertant colonies in the titre plates divided by the toxicity test plates was evaluated to determine cytoxicity. If the quotient is below 2, the test item is considered as non-cytotoxic.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain is observed. A concentration-related increase over the range tested is also taken as a sign of mutagenicity.

Validity criteria
Negative control plates must demonstrate the characteristic mean number of spontaneous revertants compared to historical control data.

Statistics:

Mean values and standard errors were calculated from the examined parameters.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was tested at 5007 µg/plate and 1504 µg/plate with 4 plates per strain on maximal soft agar. The quotient titre/tox (number of colonies in medium/number of colonies in test solutions) was calculated below 2 (see table 3). Thus, the test item was considered not to induce cytotoxicity.

Any other information on results incl. tables

Acceptability of the study

Nearly all spontaneous revertants and all positive control values were within the range of historical data. Difference of revertants lying outside the range in the first experiment are marginal. In detail, the mean number of revertants for TA 97a in the water control plates counted 97 ± 3.4 which is slightly below the historical range reaching from 99 - 159 ± 11.

Additionally, the mean number of revertants for TA 100 in DMSO control plates counted 88 ± 4.6 which is slightly below the historical range from 89 - 155 ± 14.Therefore, the study was considered as valid.

 

 

Table 1. Experiment 1: Test results for D-PrisM (plate incorporation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± standard deviation
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	TA 97a	TA 98	TA 102
–	DMSO	88± 4.6	16 ± 1.3	108± 2.5	10± 2.4	171± 8.8
–	H2O	105± 5.3	22 ± 4.1	97± 3.4	11± 2.7	151± 24.4
–	55	94± 18	15± 3	105± 10	14± 1	131± 27
–	150	96± 4	16± 2	110± 5	15± 2	125± 19
–	499	111± 4	17± 3	112± 5	10± 1	127± 22
–	1508	94± 15	15 ± 2	111± 6	10± 2	133± 17
–	5005	104± 7	14 ± 2	106± 9	13± 3	140± 8
–	Positive controls	SA	SA	4NPD	4NPD	4NPD
	Mean No. of colonies/plate ± SD	495 ± 60	201 ± 18	456 ± 18	240 ± 19	604±99
+ RLI (4%)	DMSO	112± 8.8	14± 1.9	115± 1.7	14± 3.2	171± 4.1
+ RLI (4%)	H2O	128 ± 18.7	15 ± 2.1	108 ± 8.0	13 ± 6.2	176 ± 16.8
+ RLI(4%)	55	105 ± 15	10 ± 3	99 ± 6	15 ± 4	145 ± 17
+ RLI(4%)	150	105 ± 10	11 ± 2	111 ± 5	14 ± 2	128 ± 10
+ RLI(4%)	499	98 ± 8	11 ± 1	110 ± 5	14 ± 4	126 ± 23
+ RLI(4%)	1508	111 ± 4	14 ± 3	114 ± 5	12 ± 3	150 ± 5
+ RLI(4%)	5005	98 ± 16	16 ± 2	108 ± 6	12 ± 2	132 ± 18
+ RLI(4%)	Positive controls	2AA	2AA	2AA	BaP	2AA
	Mean No. of colonies/plate ± SD	446 ± 63	201 ± 11	499 ± 22	202 ± 13	594 ± 88

SA = Sodium azide

4NPD = 4-Nitro-o-phenylenediamine

2AA = 2-Amino-anthracene

BaP = Benzo-a-pyrene

SD = standard deviation

RLI = induced male Sprague Dawley rat liver S9

 

Table 2. Experiment 2: Test results for D-PrisM (preincubation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± standard deviation
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	TA 97a	TA 98	TA 102
–	DMSO	92± 5.1	12± 1.7	102± 4.3	15± 1.7	147± 8.3
–	H2O	100 ± 7.5	14± 2.9	118± 2.2	16± 2.8	178± 20.3
–	324	95± 6	13± 2	117± 6	16± 3	168± 17
–	625	81± 14	14± 2	113± 2	14± 1	180± 24
–	1247	76± 11	14± 2	102± 5	13± 1	161± 3
–	2527	102± 17	16± 2	116± 3	15± 2	171± 14
–	5003	100± 16	16±2	114± 4	14± 3	147± 4
–	Positive controls	SA	SA	4NPD	4NPD	4NPD
	Mean No. of colonies/plate ± SD	513± 38	230± 25	475± 68	221± 15	635 ± 51
+ RLI (4%)	DMSO	88 ± 11.8	11 ± 2.2	110 ± 2.2	14 ± 2.9	159 ± 8.7
+ RLI (4%)	H2O	86± 8.5	13± 2.2	112± 5.5	15± 3.9	155± 8.7
+ RLI(4%)	324	83± 5	12± 4	110± 7	13± 1	154± 10
+ RLI(4%)	625	79± 5	12± 2	111± 2	14± 1	191± 9
+ RLI(4%)	1247	89± 17	9± 3	114± 5	13± 3	175± 5
+ RLI(4%)	2527	91± 11	13± 1	116± 7	13± 3	147± 3
+ RLI(4%)	5003	86± 6	11± 3	107± 4	13± 3	153± 16
+ RLI(4%)	Positive controls	2AA	2AA	2AA	BaP	2AA
	Mean No. of colonies/plate ± SD	472 ± 144	217 ± 15	554 ± 23	242 ± 22	612 ± 72

SA = Sodium azide

4NPD = 4-Nitro-o-phenylenediamine

2AA = 2-Amino-anthracene

BaP = Benzo-a-pyrene

SD = standard deviation

RLI = induced male Sprague Dawley rat liver S9

 

Table 3: Toxicity control

Titre values		TA 97a		TA 98		TA 100		TA 102		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
		280± 8	316± 8	208± 37	213± 27	238± 11	271± 13	273± 58	220± 14	302± 99.0	277± 63.6
Toxicity control	1504 µg/mL	274± 17.0	267± 49.5	258± 5.7	275± 7.1	317± 35.4	276± 49.5	277± 52.3	336± 19.8	275± 4.2	247± 21.2
	5007 µg/mL	237± 7.1	248± 2.8	186± 5.7	209± 4.2	239± 4.2	250± 5.7	334± 11.3	358± 5.7	237± 7.1	223± 15.6
Titre/Tox	1504 µg/mL	1.02	0.59	0.81	0.39	0.75	0.51	0.99	0.33	1.10	0.56
	5007 µg/mL	1.18	0.64	1.12	0.51	1.0	0.54	0.82	0.31	1.27	0.62

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative